25.6.13

Date: 25.6.13
Time: 19:49
Location: St. Edmund Hall, Oxford, England

This morning began with some work with Helen transfecting cells.  This is done, like before, by preparing and separating out DNA samples that are then put back into the cells by blowing holes in them using electrocution.  This was very exciting as I personally was able to electrocute the cells!!

The cells are then put on microscope slides.  This is a very lengthy process and therefore took most of the morning.  We then looked at the cells under the microscope and found some good images where the protein of interest was present and well-indicated.

Next, Mike gave me an overview of mitochondrial DNA (which is referred to as the kinetoplast in trypanosomes).  In human cells, there are tons of mitochondria within each cell, however trypanosomes are different.  They only have one kinetoplast.  It is believed that mitochondria were independent species such as bacteria that were floating around near the cells and one day the cells absorbed the mitochondria.

Through evolution now every cell has mitochondria through cell splitting, however the mitochondria have their own DNA, which is independent from the cell.  Mitochondrial DNA comes from the maternal side so apparently you can trace the entire human species back to seven or so women.  This also means, unfortunately, that you cannot get away with murder if only your mitochondrial DNA was left at the scene (on the off chance of that happening) because they would only have to go to your mother, grandmother, or any siblings with the same mother to get a 100% match, which would actually be easier than using any family member for a typical DNA match, as the match would not be 100% since half the DNA is from the mother and half from the father.  Compared to nuclear DNA, there is much more mitochondrial DNA.  This makes it the preferred identification method for large disasters with great destruction because there is a much greater chance of finding usable DNA.  Trees also utilize mitochondria in their chlorophyll, which are used for photosynthesis.  Trees absorb carbon dioxide which they then turn into glucose.  In a separate process, water that the tree absorbs is turned into oxygen and expelled from the tree.  (So if anyone has ever said trees turn CO2 into O2, they are technically incorrect).  The mitochondria and cell, which used to be separate entities, combined to move the glucose and water through the cell more efficiently.  Also, another fun fact, when it gets dark out, trees respire (as in they breathe the oxygen they produce).

After learning about the wonders of the kinetoplast, I was able to observe Jack perform a Western blot (which I also watched on the first day but now it makes much more sense).  Western blots work sort of like what Sam was doing the other day with DNA but instead with proteins.  The samples are placed in wells and are dyed.

Antibody Tagging in a Western Blot

 A charge is then run through the system and the negatively charged proteins move toward the positive charge.  As the proteins move, they encounter friction, meaning the smaller proteins are able to move farther toward the positive charge whereas the larger proteins do not move as far, staying closer to their starting point.  While the proteins are being run through with the current, six pieces of paper are being soaked in a buffer solution.  Once the proteins are finished, a membrane, which has been bathed in milk for some time to cover the surface with proteins from the milk so that when the antibodies are added they do not just stick to the membrane (which is very sticky), is put alongside the paper with the proteins.  This pair is surrounded with the six pieces of paper soaked in buffer (three to a side).  Another current is run through the system, however through as opposed to across this time.

Protein transferring to the membrane (I learned about cathodes
and anodes in chem!)

 The proteins, which are still negative, move from the paper to the membrane towards the positive charge.  Once transferred, the membrane with film is brought to the x-ray developer where the spots on the membrane are transferred to x-ray film, which is then developed.  The time which the membrane spends next to the film before being developed can differ, with the longer the time the membrane is next to the film before developing, the darker the spots once developed.  Therefore, you might think it would be better to just leave the membrane next to the film as long as possible in order to ensure all the proteins were identified as clearly as possible, however this is not the case.  In developing the film you can also determine the concentration of the proteins present.  Leaving the membrane next to the film for too long will saturate the film and prevent you from being able to determine how much protein is in the sample.